Morboeuf O, Borgel D, Aiach M, Kaabache T, Gandrille S, Gaussem P
Unité INSERM 428, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris V, 4 avenue de l'Observatoire, 75270 06,., Paris cedex, France.
Thromb Res. 2000 Oct 1;100(1):81-8. doi: 10.1016/s0049-3848(00)00296-6.
To characterize the putative biochemical modifications induced by the Ser 460 to Pro (Heerlen) mutation in protein S (PS), we expressed both wild-type (wt) and mutated recombinant PS in HEK cells. In SDS-polyacrylamide gels, r-PS Heerlen migrated at 71 kDa whereas r-wt PS migrated at 73 kDa, a difference abolished after deglycosylation by N-glycosidase, suggesting that the Ser 460 Pro mutation abolishes N-glycosylation of Asn 458. The affinity of r-wt PS and r-PS Heerlen for C4b-binding protein (C4b-BP) and for phospholipid vesicles was similar. Neither the enhancement of APC-dependent prolongation of the APTT, nor the specific enhancement of FVa and FVIIIa proteolysis by APC in purified systems was affected by the mutation. However, the Ser 460 Pro mutation induced a slight conformational change in the SHBG domain of the PS molecule, as shown by reduced binding affinity for monoclonal antibodies. The type III phenotype associated with the Heerlen mutation might thus result from a slightly modified rate of synthesis or catabolism. The resulting moderate decrease in the circulating PS concentration may modify the equilibrium between free PS and C4b-BP/PS complexes.
为了表征蛋白质S（PS）中Ser 460突变为Pro（Heerlen）所诱导的假定生化修饰，我们在HEK细胞中表达了野生型（wt）和突变型重组PS。在SDS-聚丙烯酰胺凝胶中，r-PS Heerlen在71 kDa处迁移，而r-wt PS在73 kDa处迁移，经N-糖苷酶去糖基化后这种差异消失，这表明Ser 460 Pro突变消除了Asn 458的N-糖基化。r-wt PS和r-PS Heerlen对C4b结合蛋白（C4b-BP）和磷脂囊泡的亲和力相似。在纯化系统中，该突变既不影响活化部分凝血活酶时间（APTT）依赖于活化蛋白C（APC）的延长增强，也不影响APC对FVa和FVIIIa蛋白水解的特异性增强。然而，Ser 460 Pro突变在PS分子的性激素结合球蛋白（SHBG）结构域诱导了轻微的构象变化，这通过对单克隆抗体的结合亲和力降低得以体现。因此，与Heerlen突变相关的III型表型可能是由合成或分解代谢速率的轻微改变导致的。循环中PS浓度的适度降低可能会改变游离PS与C4b-BP/PS复合物之间的平衡。
